A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV) (2)

Assay Rationale and Summary: Currently, there are no commercially available vaccines to protect humans against Respiratory syncytial virus (RSV). RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Nevertheless, severe lower respiratory tract disease may occur at any age, more ..

Assay Rationale and Summary: Currently, there are no commercially available vaccines to protect humans against Respiratory syncytial virus (RSV). RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Nevertheless, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. The existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis(R) from MedImmune) that is limited to use in high risk pediatric patients. The economic impact of RSV infections due to hospitalizations and indirect medical costs is greater than $ 650 million annually. The assay provider has developed and validated a time of addition assay to determine the point in the RSV lifecycle that the DR/cytotoxicity assay hits inhibited. This screen ascertains if the inhibition activity of the compound was early (entry) or late (replication) in the virus life cycle using a luminescent-based detection system for signal endpoint. We anticipate that the proposed studies utilizing the Molecular Libraries Probes Production Network (MLPCN) HTS resources will generate multiple scaffolds targeting various junctures in the RSV viral lifecycle. These may be furthered developed into probes to construct novel single or combination therapeutics.

Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in Optimem 1 with 2 mM L-glutamine and 5% fetal bovine serum (FBS) at 37 degrees Celsius in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.25% trypsin-EDTA. Assay Media - Preparation of Complete DMEM/F12: 50 mL Pen/Strep/Glutamine (Gibco, Cat. No. 10378) was added to four liters of room temperature DMEM/F12 (Sigma, Cat. No. D6434) and the pH adjusted to 7.5 using 1N NaOH. The medium was sterile filtered through a 0.2 um filter and 10 mL of HI-FBS was added per 500 mL of media.Compound Preparation: For time of addition assays, compounds or carrier control (DMSO) were diluted to 80x in Complete DMEM/F12 and 10 micro-liters was dispensed to assay plates (final plate well concentration was 25 micro-molar and a final DMSO concentration of 0.5%).Control Drug: The positive control drug for this assay, ribavirin [1] (No. 196066, MP Biomedicals, Solon, OH) was solubilized in DMSO. It was diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 35 micro-molar. All wells contained 0.5% DMSO.Preparation of HEp-2 cells: Cells were harvested and resuspended to 80,000 cells per ml in Complete DMEM/F12.Assay Set up: HEp-2 cells were plated in 96 well black tissue culture plates at 10,000 cells per well in 80 microliters and incubated 24 hours at 37 degrees Celsius, 5% CO2. All cell plating was conducted using a Matrix WellMate and cells were maintained at room temperature with stirring during the plating process. Compounds were diluted in media to give a final concentration of 25 micro-molar and added to plates at -1, 0, 1, 4, 6, 20 and 24 hours post-infection (p.i.). The cells were infected with RSV Long strain at an MOI of 0.12 and the assay plates were incubated for six days at 37 degrees C, 5% CO2 and 90% relative humidity. Endpoint Read: Following the six day incubation period, the assay plates were equilibrated to room temperature for 20 min and an equal volume (100 micro-liters) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a WellMate (Matrix, Hudson, NH) and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer EnvisionTM multi-label reader (PerkinElmer, Wellesley, MA) with an integration time of 0.1 s. Data Analysis: The percentage of viable cells was calculated for each of the compound addition time points to compare the effect of earlier to that of later stage addition.

Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.All three compounds showed activity (greater than 60% cell viability) up to 3 h post-infection. This early, time-limited efficacy is likely due to the inhibition of one or more early virus life cycle steps (entry, post-entry, or early-stage infection processes), and this hypothesis is supported by an inability to affect processes later in the viral replication cycle. This indicates that the probe is likely an inhibitory of early infection events (characterized by viral attachment, uptake, fusion or initial transcription).Compounds with a returned value of >60% cell viability at any timepoint of compound addition as "active". The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score . Compounds in the primary screen are scored on a scale of 0-40 based on inhibitory activity where a score of 40 corresponds to 100% inhibition. In the initial confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the IC50 result while compounds that did not confirm as actives were given the score of 0. In assays using purified and synthesized compounds a scale of 81-100 . Active compounds in this dataset are scored based on the maximum % cell viability observed across all compound addition timepoints. Compounds that did not confirm activity were given the score of 0.